DESCRIPTION: The biological function of RNA is intimately related to its three-dimensional structure. How an RNA sequence specifies its tertiary structure is an ultimate goal of RNA structural biology. In order to understand how RNA folding is accomplished, it is essential to elucidate the thermodynamic stability of the RNA structure and the kinetic folding pathway. The long-term objective of this proposal is to determine the thermodynamics and the folding kinetics of two highly structured RNAs. In the first two aims, the ribozyme component from bacterial RNase P will be studied to understand folding of a large RNA containing multiple domains. The compactness and tertiary structure of two known thermodynamic/kinetic intermediates will be evaluated. The role of the domain-domain interactions in folding will be examined. Folding will also be studied during transcription under simulated cellular conditions to determine how large RNAs may fold in vivo. In addition, RNase P RNA variants will be isolated, whose folding pathway may be altered from that of the wild-type ribozyme. In the third aim, transfer RNA will be studied as a model system to understand folding of a single, isolated tertiary structure. The tRNA studies will not only elucidate how tRNAs fold, but also more importantly, generate a conceptual framework in physico-chemical analysis for folding of all RNAs. The results from this proposal will deepen our understanding in how an RNA folds into a particular tertiary structure and establish the fundamental principles of RNA folding.